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Microglia cells are the immune effector in the Central Nervous System (CNS). However, studies have showed that they contribute more to glioma progression than to its elimination. Rutin and its aglycone quercetin are flavonoids present in many fruits as well as plants and have been demonstrated to bear anti-inflammatory, antioxidant and antitumor properties also to human glioblastoma cell lines. Previous studies also demonstrated that rutin, isolated from the Brazilian plant Dimorphandra mollis Bent., presents immunomodulatory effect on astrocytes and microglia. In this study, we investigate the antitumor and immunomodulatory properties of rutin and its aglycone quercetin on the viability of glioma cells alone and under direct and indirect interaction with microglia. Flavonoid treatment of rat C6 glioma cells induced inhibition of proliferation and migration, and also induced microglia chemotaxis that was associated to the up regulation of tumor necrosis factor (TNF) and the down regulation of Interleukin 10 (IL-10) at protein and mRNA expression levels, regulation of mRNA expression for chemokines CCL2, CCL5 and CX3CL1, and Heparin Binding Growth Factor (HDGF), Insulin-like growth factor (IGF) and Glial cell-derived neurotrophic factor (GDNF) growth factors. Treatment of human U251 and TG1 glioblastoma cells with both flavonoids also modulated negatively the expression of mRNA for IL-6 and IL-10 and positively the expression of mRNA for TNF characterizing changes to the immune regulatory profile. Treatment of microglia and C6 cells either in co-cultures or during indirect interaction, via conditioned media from glioma cells treated with flavonoids or via conditioned media from microglia treated with flavonoids reduced proliferation and migration of glioma cells. It also directed microglia towards an inflammatory profile with increased expression of mRNA for IL-1β, IL-6, IL-18 and decreased expression of mRNA for nitric oxide synthase 2 (NOS2) and prostaglandin-endoperoxide synthase 2 (PTGS2), arginase and transforming growth factor beta (TGF-β), as well as Insulin-like growth factor (IGF). Treatment of U251 cells with flavonoids also reduced tumorigenesis when the cells were xenotransplanted in rat brains, and directed microglia and also astrocytes in the microenvironment of tumor cell implantation as well as in the brain parenchyma to a not favorable molecular inflammatory profile to the glioma growth, as observed in cultures. Together these results demonstrate that the flavonoid rutin and its aglycone quercetin present antiglioma effects related to the property of modulating the microglial inflammatory profile and may be considered for molecular and preclinical studies as adjuvant molecules for treatment of gliomas.

Astrocytes preserve the brain microenvironment homeostasis in order to protect other brain cells, mainly neurons, against damages. Glial cells have specific functions that are important in the context of neuronal survival in different models of central nervous system (CNS) diseases. Microglia are among these cells, secreting several molecules that can modulate astrocyte functions. Although 1,2-dihydroxybenzene (catechol) is a neurotoxic monoaromatic compound of exogenous origin, several endogenous molecules also present the catechol group. This study compared two methods to obtain astrocyte-enriched cultures from newborn Wistar rats of both sexes. In the first technique (P1), microglial cells began to be removed early 48 h after primary mixed glial cultures were plated. In the second one (P2), microglial cells were late removed 7 to 10 days after plating. Both cultures were exposed to catechol for 72 h. Catechol was more cytotoxic to P1 cultures than to P2, decreasing cellularity and changing the cell morphology. Microglial-conditioned medium (MCM) protected P1 cultures and inhibited the catechol autoxidation. P2 cultures, as well as P1 in the presence of 20% MCM, presented long, dense, and fibrillary processes positive for glial fibrillary acidic protein, which retracted the cytoplasm when exposed to catechol. The Ngf and Il1beta transcription increased in P1, meanwhile astrocytes expressed more Il10 in P2. Catechol decreased Bdnf and Il10 in P2 cultures, and it decreased the expression of Il1beta in both conditions. A prolonged contact with microglia before isolation of astrocyte-enriched cultures modifies astrocyte functions and morphology, protecting these cells against catechol-induced cytotoxicity.

Traumatic brain injury (TBI) is a critical health problem worldwide, with a high incidence rate and potentially severe long-term consequences. Depending on the level of mechanical stress, astrocytes react with complex morphological and functional changes known as reactive astrogliosis. In cases of severe tissue injury, astrocytes proliferate in the area immediately adjacent to the lesion to form the glial scar, which is a major barrier to neuronal regeneration in the central nervous system. The flavonoid agathisflavone has been shown to have neuroprotective, neurogenic, and immunomodulatory effects and could have beneficial effects in situations of TBI. In this study, we investigated the effects of agathisflavone on modulating the responses of astrocytes and neurons to injury, using the in vitro scratch wound model of TBI in primary cultures of rat cerebral cortex. In control conditions, the scratch wound induced an astroglial injury response, characterized by upregulation of glial fibrillary acidic protein (GFAP) and hypertrophy, together with the reduction in proportion of neurons within the lesion site. Treatment with agathisflavone (1 μM) decreased astroglial GFAP expression and hypertrophy and induced an increase in the number of neurons and neurite outgrowth into the lesion site. Agathisflavone also induced increased expression of the neurotrophic factors NGF and GDNF, which are associated with the neuroprotective profile of glial cells. These results demonstrate that in an in vitro model of TBI, the flavonoid agathisflavone modulates the astrocytic injury response and glial scar formation, stimulating neural recomposition.

This study aimed to investigate the antitumor and immunomodulatory properties of the flavonoid apigenin (5,7,4'-trihydroxyflavone), which was extracted from Croton betulaster Mull, in glioma cell culture using the high-proliferative rat C6 glioma cell line as a model. Apigenin was found to have the ability to reduce the viability and proliferation of C6 cells in a time-dependent and dose-dependent manner, with an IC50 of 22.8 µmol/l, 40 times lower than that of temozolomide (1000 µmol/l), after 72 h of apigenin treatment. Even after C6 cells were treated with apigenin for 48 h, high proportions of C6 cells entered apoptosis (39.56%) and autophagy (22%) as shown by flow cytometry using annexin V/propidium iodide and acridine orange staining, respectively. In addition, the flavonoid apigenin induced cell accumulation in the G0/G1 phase of the cell cycle and inhibited glioma cell migration efficiently. Moreover, apigenin induced astroglial differentiation and morphological changes in C6 cells, characterized by increased expression of glial fibrillary acidic protein and decreased expression of nestin protein, a typical marker of neuronal precursors. The immunomodulating effects of apigenin were also characterized by a change in the inflammatory profile as evidenced by a significant decrease in interleukin-10 and tumor necrosis factor production and increased nitric oxide levels. Because apigenin can induce differentiation, apoptosis, and autophagy, can alter the profile of cytokines involved in regulating the immune response, and can reduce the survival, growth, proliferation, and migration of C6 cells, this flavonoid may be considered a potential antitumor drug for the adjuvant treatment of malignant gliomas.

Rutin is a glycosylated flavonoid present in many fruits and plants that has been demonstrated to have anti-inflammatory and antioxidant properties. However, little is known about the mechanisms underlying microglial activation and its effects on the regulation of cytokines and chemokines associated with inflammatory responses in the central nervous system. In this study we examined the effect of rutin on resting or lipopolysaccharide (LPS)-stimulated microglia and characterized their modulation to an activated M1 phenotype or an alternatively activated M2 phenotype. Microglial cells were treated with rutin (1–100 μM); alternatively, microglial cells were stimulated with LPS and the cells were then treated with rutin (50 μM). The results revealed that rutin treatment was not toxic to microglial cells and induced a dose-dependent increase in microglial proliferation associated with changes in morphology after 24 h of treatment. Rutin also induced microglial activation characterized by an increase in OX-42 positive cells and a large proportion of cells with a CD150/CD206-positive M2 phenotype. Rutin also induced a decrease in the mRNA levels of TNF, IL1β, IL6 and iNOS, reduced the production of IL6, TNF, and nitric oxide, and increased production of the M2 regulatory cytokine IL10 and arginase. Rutin also significantly inhibited the LPS-induced expression of PTGS2, IL18 and TGFβ mRNA. These findings show that rutin has the ability to promote microglial proliferation and induces microglial polarization to the M2 profile when cells are stimulated with LPS. These results point this flavonoid as a possible alternative in the treatment or prevention of neurodegenerative disorders.

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly of the Th1 type. However, infected primary glial cell cultures release IL-10 and IL-6 but not IFN-γ. This suggests a rather protective response of the glia to avoid inflammatory damage of the nervous tissue. In this study, we investigated the effects of pro-inflammatory cytokines in primary mixed cultures of rat astrocytes and microglia infected with N. caninum. The cells were treated with either IFN-γ, TNF-α, anti-IL-10 or anti-TGF-β antibodies and were infected with parasite tachyzoites 24h later. Trypan Blue exclusion and MTT assays were performed to test cell viability. It was observed that cytokines, antibody treatment and in vitro infection did not reveal significant cell death in the various culture conditions. Treatment with 50, 150 and 300 IU/mL of either IFN-γ or TNF-α reduced tachyzoites numbers in cultures by 36.7%, 54.8% and 63.8% for IFN-γ and by 27.6%, 38.4% and 29.7% for TNF-α, respectively. In the absence of IL-10 and TGF-β, tachyzoite numbers were reduced by 52.8% and 41.5%, respectively. While IFN-γ (150 and 300 IU/mL) increased the nitrite levels in uninfected cells, parasite infection seemed to reduce the nitrite levels, and this reduction was more expressive in IFN-γ-infected cells, thereby suggesting an inhibitory effect on its production. However, TNF-α, IL-10 and TGF-β did not affect the nitrite levels. Basal PGE(2) levels also increased by 17% and 25%; 78% and 13% in uninfected and infected cells treated with IFN-γ or anti-TGF-β, respectively. Nevertheless, the antibody neutralization of IL-10 reduced PGE(2) release significantly. These results highlight the possibility of a combined effect between the IFN-γ and parasite evasion strategies and show that the IFN-γ, TNF-α, IL-10 and TGF-β cytokines participate in parasite proliferation control mechanisms.

Studies have shown cases of poisoning with plants from the genus Crotalaria (Leguminosae) mainly in animals. They induce damages in the central nervous system (CNS), which has been attributed to toxic effects of the pyrrolizidine alkaloid (PA) monocrotaline (MCT). Previously we demonstrated that both MCT and dehydromonocrotaline (DHMC), its main active metabolite, induce changes in the levels and patterns of expression of the main protein from astrocyte cytoskeleton, glial fibrillary acidic protein (GFAP). In this study we investigated the effect of MCT on rat cortical astrocyte/neuron primary co-cultures. Primary cultures were exposed to 10 or 100 μM MCT. The MTT test and the measurement of LDH activity on the culture medium revealed that after 24h exposure MCT was not cytotoxic to neuron/astrocyte cells. However, the cell viability after 72 h treatment decreased in 10-20%, and the LDH levels in the culture medium increased at a rate of 12% and 23%, in cultures exposed to 10 or 100 μM MCT. Rosenfeld staining showed vacuolization and increase in cell body in astrocytes after MCT exposure. Immunocytochemistry and Western blot analyses revealed changes on pattern of GFAP and βIII-tubulin expression and steady state levels after MCT treatment, with a dose and time dependent intense down regulation and depolarization of neuronal βIII-tubulin. Moreover, treatment with 100 μM MCT for 12h induced GSH depletion, which was not seen when cytochrome P450 enzyme system was inhibited indicating that it is involved in MCT induced cytotoxicity in CNS cells.

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-alpha, IFN-gamma, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-gamma was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8+/-0.6 and 3.7+/-0.6 pg TNF-alpha/mg protein and 2.7+/-0.69 and 4.1+/-0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24h post-infection (2.3+/-0.8 pg/mg protein) until 72 h (4.2+/-1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-gamma; high levels of TNF-alpha and NO may represent an effective response by infected glial cells against N. caninum.

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 micromol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 micromol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 micromol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld's staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 micromol/L rutin. Enhanced TNFalpha release was observed in astrocyte primary cultures treated with 100 micromol/L rutin and in mixed primary cultures treated with 50 and 100 micromol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFalpha production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.

Rutin is a flavonoid obtained from Dimorphandra mollis (Benth.), a medicinal Brazilian plant used as antioxidative, antihemorrhagic, and blood vessel protector. The present study has examined its effects on the viability and function of immune system cells in vitro. Rat spleen and thymus cells were cultured with 10 nM, 1 microM, and 10 microM of the drug in the presence or absence of PWM, LPS, or ConA mitogens. Cellular proliferation was analyzed by H(3)-thymidin uptake and IFN-gamma and IL-10 were measured by ELISA after 48 and 72 hr. Viability was measured by flow cytometry using Annexin V and PI after 24 and 48 hr. The flavonoid rutin inhibited splenocytes and thymocytes proliferation under ConA stimulation observed by an increase on apoptosis levels of thymocytes stimulated with PWM in 24 hr and on splenocytes stimulated with PWM in 48 hr. Function studies showed a decrease on IFN-gamma production by splenocytes and thymocytes stimulated with PWM or ConA. Spleen cells cultured with LPS and rutin showed a decrease on apoptosis after 24 hr and an increase on the IL-10 levels after 48 hr. There was no significant variation on the necrosis rate, viability, and function of cells treated with rutin in the absence of mitogenic stimulus.

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.

Dehydromonocrotaline (DHMC) is the main monocrotaline active cytochrome P450's metabolite, and has already been assessed in the CNS of experimentally intoxicated rats. DHMC effects were here investigated toward rat astroglial primary cultures regarding cytotoxicity, morphological changes and regulation of GFAP expression. Cells, grown in DMEM supplemented medium, were treated with 0.1-500 microM DHMC, during 24- and 72-h. According to MTT and LDH tests, DHMC was toxic to astrocytes after 24-h exposure at 1 microM, and induced membrane damages at 500 microM. Rosenfeld dying showed hypertrophic astrocytes after 72-h exposure to 0.1-1 microM DHMC. GFAP immunocytochemistry and western immunoblot revealed an increase of GFAP labelling and expression, suggesting an astrogliotic reaction to low concentrations of DHMC. At higher concentrations (10-500 microM), astrocytes shrank their bodies and retracted their processes, presenting a more polygonal phenotype and a weaker expression on GFAP labelling Nuclear chromatin staining by Hoechst-33258 dye, revealed condensed and fragmented chromatin in an important proportion (+/-30%) of the astrocytes exposed to 100-500 microM DHMC, suggesting signs of apoptosis. Our results confirm a cytotoxic and dose-dependent effect of DHMC on cultures of rat cortical astrocytes, leading to apoptotic figures. These effects might be related to the neurological damages and clinical signs observed in animals intoxicated by Crotalaria.

Prosopis juliflora is used for feeding cattle and humans. Intoxication with the plant has been reported, and is characterized by neuromuscular alterations and gliosis. Total alkaloidal extract (TAE) was obtained using acid/basic-modified extraction and was fractionated. TAE and seven alkaloidal fractions, at concentrations ranging 0.03-30 microg/ml, were tested for 24h on astrocyte primary cultures derived from the cortex of newborn Wistar rats. The MTT test and the measure of LDH activity on the culture medium, revealed that TAE and fractions F29/30, F31/33, F32 and F34/35 were cytotoxic to astrocytes. The EC(50) values for the most toxic compounds, TAE, F31/33 and F32 were 2.87 2.82 and 3.01 microg/ml, respectively. Morphological changes and glial cells activation were investigated through Rosenfeld's staining, by immunocytochemistry for the protein OX-42, specific of activated microglia, by immunocytochemistry and western immunoblot for GFAP, the marker of reactive and mature astrocytes, and by the production of nitric oxide (NO). We observed that astrocytes exposed to 3 microg/ml TAE, F29/30 or F31/33 developed compact cell body with many processes overexpressing GFAP. Treatment with 30 microg/ml TAE and fractions, induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. We also observed that when compared with the control (+/-1.34%), the proportion of OX-42 positive cells was increased in cultures treated with 30 microg/ml TAE or F29/30, F31/33, F32 and F34/35, with values raging from 7.27% to 28.74%. Moreover, incubation with 3 microg/ml F32, 30 microg/ml TAE, F29/30, F31/33 or F34/35 induced accumulation of nitrite in culture medium indicating induction of NO production. Taken together these results show that TAE and fractionated alkaloids from P. juliflora act directly on glial cells, inducing activation and/or cytotoxicity, stimulating NO production, and may have an impact on neuronal damages observed on intoxicated animals.

Texto completo: acesso restrito. p.193–197 Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-08-29T16:08:01Z No. of bitstreams: 1 1-s2.0-S0014489405002791-main.pdf: 301544 bytes, checksum: e6fd21c4145ac6f1edcc75fe0a4c36dc (MD5) Approved for entry into archive by Patricia Barroso(pbarroso@ufba.br) on 2013-09-02T15:24:40Z (GMT) No. of bitstreams: 1 1-s2.0-S0014489405002791-main.pdf: 301544 bytes, checksum: e6fd21c4145ac6f1edcc75fe0a4c36dc (MD5) Made available in DSpace on 2013-09-02T15:24:40Z (GMT). No. of bitstreams: 1 1-s2.0-S0014489405002791-main.pdf: 301544 bytes, checksum: e6fd21c4145ac6f1edcc75fe0a4c36dc (MD5) Previous issue date: 2006 The effect of Neospora caninum, a parasite that causes abortion and neuromuscular changes, has been investigated on a major population of neural cells, the astrocytes. Highly enriched astroglial primary cultures obtained from neonatal rats were infected after 21 days of culture. Astroglial reactivity, IL-10 and IFN-γ expression, and cell viability (lactate dehydrogenase activity, metabolization of tetrazolium salt, and trypan blue exclusion assay) have been investigated after 24 and 72 h of infection. Astroglial hypertrophy, gliofilament reorganization, metabolic changes suggesting hypoxia and a strong IL-10 release have been observed in the infected cells. These results show that neural cells are targets for the parasite and that astrocytes may contribute to the CNS immune response to the parasite.

The protozoan Neospora caninum has a veterinary importance because it causes abortion in cattle and neuromuscular alterations in dogs. We infected rat astrocytes, in vitro , with different concentrations of N. caninum . Astrocytes responded to infection by producing the pro-inflammatory cytokine TNF-α and the neurotoxic—free radical NO, 24 and 72 h post-infection. These data suggest that astrocytes, which are essential for brain function, are targets for the parasite and this represents a practical and valid model to study the effects of N. caninum on the CNS.